Cassette for electrophoretic gels

ABSTRACT

An improved cassette for use in the formation of an electrophoretic gel comprises two plates with substantially planar walls having two sides and two ends so arranged in a side by side spaced apart array to form a gel receiving space between them. A plurality of dividing ribs on one or each of the walls extend from a first end of the wall or walls substantially parallel to at least one of the sides thereof to a rib base end. The ribs extend into the space so as to subdivide at least one end of the space into a plurality of substantially parallel wells. A plurality of holes extend through at least one of the walls of the cassette located at or adjacent the base of the dividing rib and aligned with the rib. The arrangement of holes allow sufficient current flow to replace that lost due to the dividing ribs. The structure retains the advantage of having solid dividing walls which are considerably more resilient than dividing walls made from fingers of gel even when reinforced with pegs or projections. and yet by virtue of the provision of a hole at the base of the ribs allows sufficient current flow at the base of each rib that the current flow through the space is substantially uniform, thus preventing spreading of the bands as they form in the body of the gel.

FIELD OF THE INVENTION

[0001] The present invention relates to a cassette for use in theformation of an electrophoretic gel and more particularly to such acassette which includes means to assist in separating and definingindividual sample receiving wells at one end of the electrophoretic gel.

BACKGROUND ART

[0002] Electrophoretic gels. usually comprising hydrogels such asagarose or polyacrylamide are used for the separation of nucleic acids.proteins and other macromolecular compounds. The sample to be separatedis placed at one end of the gel and a direct electric field is appliedbetween the ends of the gel causing the components of the sample tomigrate through the gel at rates dependent upon their molecular size andcharge.

[0003] A mixture of components to be separated is normally introducedinto one of a number of small wells formed in an upper edge of the gelbefore the electric current is applied. It is usual to run a number ofsuch mixtures simultaneously on an electrophoretic gel in a side by sidearrangement. For this purpose one mixture is placed in each of a seriesof wells formed in the upper edge of the gel.

[0004] In the past electrophoretic gels were formed by juxtaposing apair of glass plates in a slightly spaced apart side-by-siderelationship and filling the space therebetween with a liquid which canset and form an electrophoretic gel. The side edges of the space betweenthe glass plates were typically sealed with adhesive tape or a similarmaterial, and when the gel is poured a comb was placed in the upper endof the space between the glass plates. After the gel had set the combwould be withdrawn leaving a series of spaced apart wells defined in thetop of the gel. each well having been defined by one tooth of the comb.A tongue of gel remains between the glass plates separating the pair ofadjacent wells.

[0005] The gel is immersed in a buffer solution and electrodes above andbelow the gel cause a direct electric current to flow through the gel.The components in the mixtures travel through the gel from the top tothe bottom of the gel at different rates depending on their size andcharge. and separate out into bands.

[0006] In more recent years it has been proposed to preformelectrophoretic gels in cassettes formed of synthetic plasticsmaterials. The side walls of the cassette are formed with integral meansto connect them together along the sides of the cassette. Again when thegel is poured a comb is placed in the upper end of the space between thewalls of the cassette for the formation of the spaced apart wells.However, this arrangement has a disadvantage that upon withdrawal of thecomb the fingers of the gel may. with time. show an increased tendencyto break away from the remainder of the gel. This results in poorlydefined wells. Alternatively, if the tongues of gels are left intactupon withdrawal of the comb they may not firmly adhere to the plasticside wall of the cassette. This has resulted that the tongues may fallover side ways occluding an adjacent well.

[0007] One attempt to overcome this problem is described in U.S. Pat.No. 5.288,465. where ribs are provided in the cassette walls to definewells at one end of the cassette. Whilst this arrangement providesstable wells suitable to hold the sample material, this arrangement hasa disadvantage that the solid walls of the wells interfere with thesmooth flow of electric current through the electrophoretic gel: becauseof the ribs, the current is initially confined and then spreads outwhich has a result that as the run proceeds bands from adjoining wellstend to spread out and merge with one another.

[0008] International Patent Application No WO 97/04307 addresses theproblem of current interference by replacing the ribs by a plurality ofsmall projections or pegs. These projections or pegs extend into the gelto support the gel fingers. The projections support the gel whilstallowing substantially parallel flow of the current to establish beforethe current meets the samples positioned in the bottom of the wells.This parallel current flow is necessary to maintain the separationbetween the bands/samples from the different wells and prevent thesamples for spreading outwards.

[0009] However. the disadvantage of the cassette shown in WO 97/04307 isthat as the combs are withdrawn, the tops of the gel fingers tend tobreak away from the remainder of the gel finger.

[0010] The present invention is directed to alternative arrangementsaddressing the problems associated with spreading current and also theintegrity of the walls of the sample wells.

DISCLOSURE OF THE INVENTION

[0011] According to a first aspect of the present invention there isprovided an improved cassette for use in the formation of anelectrophoretic gel comprising two plates with substantially planarwalls having two sides and two ends so arranged in a side by side spacedapart array to form a gel receiving space between them, a plurality ofdividing ribs on one or each of the walls extending from a first end ofthe wall or walls substantially parallel to at least one of the sidesthereof to a rib base end and being adapted to extend into the space soas to subdivide at least one end of the space into a plurality ofsubstantially parallel wells. the improvement consisting of a pluralityof holes extending through at least one of the walls of the cassettelocated at or adjacent the base of the dividing rib and aligned with therib, the arrangement being such that the holes allow sufficient currentflow to replace that lost due to the dividing ribs.

[0012] The present invention retains the advantage of having soliddividing walls which are considerably more resilient than dividing wallsmade from fingers of gel even when reinforced with pegs or projections,and yet by virtue of the provision of a hole at the base of the ribsallows sufficient current flow at the base of each rib that the currentflow through the space is substantially uniform. thus preventingspreading of the bands as they form in the body of the gel.

[0013] The size of the gap between the sample bands can be controlled byvarying the size of the hole in the wall of the cassette. A larger holecreates a greater current flow and provides a wider gap between thesamples. The size of the hole will depend on the thickness of the riband the size of the well. The holes may vary in size between about 2 mmdiameter to 0.5 mm diameter. with a 1 mm diameter hole being typical.

[0014] It is a preferred feature that the ribs are tapered outwardlyfrom the first end to their base end so that the upper part of the wellis wider than the lower part of the well. This makes the wells easier toload with samples.

[0015] It is preferred that the sides of the holes are chamfered ortapered so that during operation the chamfered hole will fill withbuffer and not trap air bubbles.

[0016] In gel cassettes of the type described above and in theintroduction, the concentration of the gels is graduated. The cassettesare typically filled from below which means that the gel at the top ofthe cassette which is the least dense, is the least concentrated.Ironically, in cassettes of the type where gel fingers form, the wallsof the well is also the part where the gel needs to be strongest i.e.the most concentrated gel. Thus, there is a requirement for theconcentration of acrylamide in such cassettes to be high to ensure ahigh enough gel concentration for forming the fingers. However. in thepresent invention because the walls of the well are plastic and not gel,the upper zone of the gel can be a much lower concentration say around2.5% as opposed to 5% which allows the separation of a much highermolecular weight proteins having a molecular weight of around 800,000 to1,000,000.

[0017] A second aspect of the present invention provides an improvedcassette containing an electrophoretic gel comprising two plates withsubstantially planar walls having two sides and two ends so arranged ina side by side spaced apart array to form a gel receiving space betweenthem, a plurality of dividing ribs on one or each of the wall membersextending from the first end of the member or members substantiallyparallel to at least one of the sides thereof and adapted to extend intothe space so as to subdivide at least one end of the space into aplurality of substantially parallel wells having bases. the improvementcomprising that the part of the well walls formed from plastic ribmaterial does not extend to the base of the well and that the lower partof the well wall is formed from a finger of gel.

[0018] In one preferred embodiment, the finger of gel may be supportedby one or more projections.

[0019] The solid upper rib segment of the well wall resists stressesplaced on the gel fingers and maintains the integrity of the wells. Itprevents damage to the lower gel fingers defining the lower part of thewell walls when the comb is withdrawn or when samples are loaded intothe wells. Surprisingly the gel base of the wall allows there-establishment of parallel current flow through the gel before thewell base is reached by the current maintaining the separation betweenthe bands of separating molecules in the body of the gel.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The invention will now be described, by way of example only, andwith reference to the accompanying drawings in which:

[0021]FIG. 1 is a schematic front view of a cassette illustrating afirst embodiment of the present invention:

[0022]FIGS. 2a and 2 b are schematic side views illustrating currentflow through the cassette shown in FIG. 1.

[0023]FIG. 3 is a side view of a variant of the embodiment shown in FIG.1:

[0024]FIG. 4 shows a further variant of the embodiment shown in FIG. 1;and

[0025]FIG. 5 shows a second embodiment of the present invention alsoillustrating separation between bands of molecules separated byelectrophoresis.

BRIEF DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Referring to the drawings FIG. 1 shows a first electrophoresiscassette 10 which may be formed in two parts by injection moulding or byother methods from a suitable synthetic plastic material.

[0027] One part forms a first side wall 11 of the cassette which isbanded on each side by a connecting means comprising a pair of spacedapart ridges not shown.

[0028] The other part forms a second side wall 12 of the cassette whichis also banded on each side by a pair of spaced ridges. not shown. Theridges interdigitate to form a seal along each side of the cassette toprevent leakage therethrough of the gel forming liquid during setting ofthe gel or flow of an electrical current during electrophoresis.

[0029] The ridges are of a sufficient thickness that a gel receivingspace 13 is defined between the side walls which lie in substantiallyparallel planes. Such a construction as described above is already wellknown in the prior art see. for example. WO 97/04307 the contents ofwhich are incorporated herein by reference.

[0030] The inside surface of the side wall 11 is formed with a pluralityof inwardly directed parallel. spaced apart ribs 14 which extend intothe gel receiving space 13.

[0031] The ribs 14 may however originate from either or both of walls 11and 12. Each rib has a length which defines the depth of the well. and athickness which spans the gel receiving space 13.

[0032] The ribs 14 extend from a first. upper end 11 a of side wall 11to a base end 14 b which is located at the base of the well.

[0033] In an alternative embodiment, the ribs may be thicker than thegel receiving space 13 and they may mate with corresponding recessesprovided in the opposite wall 12.

[0034] At the base of each rib distal from the upper end 11 a of theside wall 11 there is a hole 15 which extends through the wall 11 and istypically around 1 mm diameter.

[0035] To form a gel in the cassette 10 a comb with a plurality ofspaced apart teeth is inserted into the upper end of the cassette. Theteeth are dimensioned so that they interdigitate with the ribs 14 butare at all times closely spaced therefrom. After the gel is formed inthe space 13 the cassette can be used.

[0036] When the comb is withdrawn. there will be a plurality of wellsformed in the gel separated by the ribs 14.

[0037] In use the cassette is inserted in a buffer solution and currentis passed through the gel via an electrode located above the upper endof the cassette and an electrode located below the lower of end of thecassette. Current flows in two paths, the first path being through thewell illustrated by arrow (A) shown in FIG. 2a and the second path (B)through the hole 15 thence through the gel to the base of the cassette.

[0038] It is that additional flow of current through the hole 15 thatprevents the current passing through the wells from spreading out andintermingling the bands.

[0039] The separation of the samples into bands of molecules isillustrated in FIG. 5. albeit in the context of a definite embodiment ofthe invention. The gap between bands can be varied by varying the sizeof the hole 15.

[0040]FIG. 3 shows a variant in which the side 16 of the hole 15 aretapered outwardly or chamfered to allow buffer to fill the hole moreeasily and to prevent air bubbles becoming trapped in the hole 15. Anair bubble would inhibit the passage of electric current through thehole defeating the object of the invention.

[0041]FIG. 4 shows a further variant on the invention in which the ribs14 are tapered being wider at their base portion adjacent the hole 15and narrower at the top portion adjacent the upper edge of side wall 11.This makes the upper part of the wells wider and makes loading the wellseasier.

[0042]FIG. 5 shows a further embodiment of the present invention inwhich the upper part of the walls of the dividing well are comprised ofplastic ribs 17 and the lower part of the walls comprise a gel finger 18which may be stabilised with one or more pegs 19 such as described in WO97/04307.

[0043] The solid upper part of the rib resists stresses placed on thefingers and maintains integrity of the walls. particularly when the combis being withdrawn or when samples are being loaded into the wells.Because the lower part of the well walls comprises a gel finger, thisallows the current to spread out sufficiently before it reaches thesamples, for separation to occur in even bands as illustrated in FIG. 4.

[0044] It will be appreciated by persons skilled in the art thatnumerous variations and/or modifications may be made to the invention asshown in the specific embodiments without departing from the spirit orscope of the invention as broadly described. The present embodimentsare, therefore, to be considered in all respects as illustrative and notrestrictive.

1. A cassette for use in the formation of an electrophoretic gelcomprising two plates with substantially planar walls having two sidesand two ends so arranged in a side by side spaced apart array to form agel receiving space between them. a plurality of dividing ribs on one oreach of the walls extending from a first end of the wall or wallssubstantially parallel to at least one of the sides thereof to a ribbase end and being adapted to extend into the space so as to subdivideat least one end of the space into a plurality of substantially parallelwells. the improvement consisting of a plurality of holes extendingthrough at least one of the walls of the cassette located at or adjacentthe base of the dividing rib and aligned with the rib, the arrangementbeing such that the holes allow additional current flow to replace thatlost due to the dividing ribs.
 2. The cassette of claim 1 wherein theplurality of holes may vary in size between about 2 mm diameter to 0.5mm diameter.
 3. The cassette of claim 1 or claim 2 wherein the holeshave a diameter of about 1 mm
 4. The cassette of claim 1 wherein theribs are tapered outwardly from an upper end to their base end so thatthe upper part of the well is wider than the lower part of the well. 5.The cassette of claim 1 wherein the sides of the holes are chamfered ortapered.
 6. An improved cassette containing an electrophoretic gelcomprising two plates with substantially planar walls having two sidesand two ends so arranged in a side by side spaced apart array to form agel receiving space between them. a plurality of dividing ribs on one oreach of the wall members extending from the first end of the member ormembers substantially parallel to at least one of the sides thereof andadapted to extend into the space so as to subdivide at least one end ofthe space into a plurality of substantially parallel wells having bases,the improvement comprising that the part of the well walls formed fromplastic rib material does not extend to the base of the well and thatthe lower part of the well wall is formed from a finger of gel.
 7. Thecassette of claim 6 wherein the finger of gel is supported by a one ormore projections.